Many procedures in medical research and recombinant DNA technology rely on the labeling of nucleotides that are then incorporated into oligo or polynucleotide probes. Commonly used labels have included radioactive isotopes, biotin, iminobiotin, haptens, fluorescent dyes and electron-dense reagents.
Problems with radioisotope labeling include the risk to the people handling the radioisotope-labeled material, and the need for elaborate and expensive safety precautions to be taken and maintained during the preparation, utilization and disposal of the radioactive material. Radioisotopes and radio-labeled nucleotides or polynucleotides are very expensive to purchase and use, due in part because of the safety precautions required and the problems in safely disposing of radioactive hazardous waste. In addition, the probe""s structural integrity and sensitivity can be rapidly degraded during storage due to radioactive decay and radiochemical decomposition.
Oligo and polynucleotides can also be labeled with biotin and iminobiotin, haptens and fluorescent dyes for the direct detection of nucleotides. For example Ward et al. in U.S. Pat. Nos. 4,711,955; 5,449,767, 5,328,824; 5,476,928; herein incorporated by reference, describe the labeling of nucleotides with a hapten, biotin, or iminobiotin. The hapten is detected by a labeled antibody and the biotin or iminobiotin is detected by a labeled avidin or strepavidin.
Ruth, U.S. Pat. No. 4,948,882, herein incorporated by reference, describes the derivatization of nucleotides with fluorescent dyes, biotin, and antigens. Again, as in Ward et al. the biotin is detected by avidin, antigens are detected by labeled antibodies, and the fluorescent dyes are directly detected by spectral techniques.
The labeled nucleotides of Ruth are incorporated into oligo or polynucleotides by conventional phosphoramidite chemistry. The synthesized oligo or polynucleotides are then used as probes to detect DNA sequences. It is important to note that these labeled nucleotides are directly detectable when the probe is used in contrast to the labeled pyrimidines of the invention.
Labeling of nucleic acid probes with fluorophores facilitates microscopic analysis of chromosomes and their genetic structure by fluorescent in situ hybridization (FISH). A method of FISH probe preparation and signal detection is described by Ward et al. In the area of DNA diagnostics, automated platforms based on labeled synthetic oligonucleotides immobilized on silicon chips work by fluorescence detection and are capable of the parallel analysis of many samples and mutations. The methods used in preparing labeled, chemically activated nucleotide precursors for oligonucleotide synthesis is discussed and demonstrated by Ruth. Nucleic acid amplification methods such as PCR have become very important in genetic analysis and the detection of trace amounts of nucleic acid from pathogenic bacteria and viruses. Analysis of many PCR reactions by standard electrophoretic methods becomes tedious, time consuming and does not readily allow for rapid and automated data acquisition. PCR has been adapted to use fluorescent molecules by incorporation of fluorescent labeled primers or nucleotides into the product which is then directly detected or by the use of fluorescent probes that are then detected. Removal of unincorporated, labeled substrates is usually necessary and can be accomplished by filtration, electrophoretic gel purification or chromatographic methods. However, the large amount of sample handling required by these analytical techniques make these purification methods labor intensive, not quantitative and they invariably leads to serious contamination problems. Affinity capture of PCR products by strepavidin coated beads or micro titer wells requires incorporation of biotin labels in addition to the fluorophores and still involves transfer steps that can lead to contamination. Instrumentation utilizing both gel electrophoresis and laser excitation optics represents an improvement in data acquisition but cannot handle large numbers of samples, retains the comparatively prolonged separation times characteristic of gels and still requires sample transfer.
The use of fluorescent energy transfer, oligonucleotide primers containing hairpin secondary structure are described in U.S. Pat. No. 5,866,336, and in pending U.S. patent application Ser. Nos.: 08/837,034 filed Apr. 11, 1997 and 08/891,516 filed Jul. 11, 1997, each of which is entitled Nucleic Acid Amplification of Oligonucleotides with Molecular Energy Transfer Labels and Methods Based Thereon. The contents of all three of these documents are herein incorporated by reference. These documents solve the background problems associated with unincorporated, labeled substrates, alleviates sample transfer problems and enables the use of a homogeneous PCR assay for the analysis of many samples without cross contamination by amplicon. The unincorporated primer contains a target-specific, single stranded region with a 3xe2x80x2-hydroxyl terminus from which polymerase catalyzed elongation occurs. Under native conditions the unincorporated primer contains a tract of self complimentary nucleotides in the 5xe2x80x2 region that are hydrogen bonded into a hairpin conformation. The 5xe2x80x2-hydroxy terminus is modified with a fluorophore. The 5xe2x80x2-deoxynucleotide is adenosine (dA). The last base of the double stranded hairpin stem region is a deoxyuridine (dU) which is base paired to the 5xe2x80x2-dA. The aromatic azo dye 4-dimethylaminoazobenzene-4xe2x80x2-sulfonyl chloride (dabsyl) is linked via a spacer arm to the C-5 carbon of the dU base. Hairpins can be extremely stable structures for their size, having high Tm""s and strongly negative free energies. A hairpin is an intramolecular formation and is much more kinetically and entropically favored than the formation of a hybridized duplex. Under these structural conditions the fluorophore and the DABSYL are tightly held in close proximity to each other. At these short molecular distances the fluorophore and DABSYL can have orbital contact and overlap, being able to form relatively weak chemical interactions such as it complexes, hydrogen bonding or salt complex formation. This orbital interaction promote very efficient transfer of excitation energy from the fluor to the dabsyl. The DABSYL acceptor is not fluorescent and dissipates much of the donated or transferred energy as heat. While resonance energy transfer plays a role in the fluorescein or other fluorophore quenching other mechanisms of energy transfer can operate over these short distances as well and can account for the very efficient quenching of the fluor by the quencher.
After target hybridization and polymerase extension, the primer becomes a template for the next round of DNA replication. Polymerase displaces the 5xe2x80x2-end of the hairpin and copies the remainder. This process opens the hairpin conformation and the primer enters into the standard double helical B DNA conformation. The fluorophore and DABSYL are then separated by more than 60 Angstroms. The incorporated primer is now capable of producing strong fluorescent emissions when exposed to the appropriate excitation wavelengths. Unincorporated primer remains as a hairpin and produces very little flourescent emission for the reasons previously stated.
The hairpin, energy transfer primers are synthesized by standard automated phosphoramidite chemistry. See Caruthers, U.S. Pat. Nos.: 4,415,734 and 4,458,066 herein incorporated by reference. Linkage of the DABSYL moiety requires post synthetic modification. During oligonucleotide synthesis a commercially available dU phosphoramidite containing an aliphatic amino group linked to the base is incorporated. After deprotection and desalting the crude oligonucleotide is purified by reverse phase HPLC followed by solvent removal. The terminal amino group of the linker is reacted with a molar excess of commercially available dabsyl-succinimidyl ester or DABSYL sulfonyl chloride under alkaline conditions for 24 to 48 hours with the periodic addition of fresh, activated, dye during the incubation. The DABSYL modified oligonucleotide is precipitated and purified by reverse phase HPLC. After solvent removal the oligonucleotide is suspended in aqueous buffer, quantitated and tested. DABSYL coupling efficiencies are typically less than 80 percent and the entire process can take over a week to make preparative amounts of material. Final yields of product are typically much less than the coupling efficiency and are usually less than 50 percent. In addition, because the incorporation of DABSYL is not quantitative and the purification of DABSYL incorporated probes is not complete the probe is contaminated with oligonucleotide having a fluorescent molecule without a DABSYL quencher. Therefore probes having a quencher incorporated by this method will have a greater background fluorescence than probes made from the quencher linked pyrimidines of the invention.
The inventors have unexpectedly discovered that when mononucleotide precursors, in particular pyrimidines, are coupled to a quencher molecule and the quencher-pyrimidine is then incorporated into a probe using conventional phosphoramidite chemistry that the quencher-pyrimidine is stable during the protection and deprotection steps and is itself incorporated in good yield during the synthesis of hairpin, energy-transfer primers. The overall synthesis of the primers is also not effected by the quencher-pyrimidine nucleotide. The fluorophore will act as a fluorescence indicator when the hairpin is unfolded because the distance between the quencher and the fluorophore is greatly increased.
Compounds having the following general structure (A) are preferred: 
wherein L is a linker group, preferably comprising at least three carbon atoms, and Q is a quencher group; P is a pyrimidine group, preferably a uracil or cytosine group, and L is linked to the carbon atom at the 4 or 5 position of P, or P is a purine group, preferably an adenine group, and L is directly or indirectly linked to the carbon at the 8 position of P; and x is an activated phosphorous group capable of bonding to a 3xe2x80x2 hydroxyl group of another nucleotide, y is a protected hydroxyl group, and z is xe2x80x94H, xe2x80x94F, xe2x80x94OCH3, xe2x80x94Oxe2x80x94allyl, a protected hydroxyl group, or a protected amine group, or y is an activated phosphorous group capable of bonding to a 5xe2x80x2 hydroxyl group of another nucleotide, x is a protected hydroxyl group, and z is xe2x80x94H, xe2x80x94F, xe2x80x94OCH3, xe2x80x94Oxe2x80x94allyl, a protected hydroxyl group, or a protected amine group.
Preferably the linker has the following structure: xe2x80x94CHxe2x95x90CHxe2x80x94C(O)xe2x80x94NHxe2x80x94(CH2)nxe2x80x94NHxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94C(O)xe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94(Oxe2x80x94CH2xe2x80x94CH2)mxe2x80x94NHxe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94C(O)xe2x80x94(CH2)nxe2x80x94NHxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94C(O)xe2x80x94NHxe2x80x94(CH2)nxe2x80x94CO2xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94C(O)xe2x80x94NHxe2x80x94(CH2)nxe2x80x94NHxe2x80x94, xe2x80x94(CH2)nxe2x80x94NHxe2x80x94, or xe2x80x94NHxe2x80x94(CH2)nxe2x80x94NHxe2x80x94, wherein n is an integer from 2 to 12, and m is 1 or 2. Most preferably, the linker has the following structure: xe2x80x94CHxe2x95x90CHxe2x80x94C(O)xe2x80x94NHxe2x80x94(CH2)6xe2x80x94NHxe2x80x94, xe2x80x94(CH2)3xe2x80x94NHxe2x80x94, or xe2x80x94NHxe2x80x94(CH2)3xe2x80x94NHxe2x80x94.
Q is a quencher capable of quenching the fluorescence of a flourescent dye when the quencher and dye are incorporated into a hairpin oligonucleotide. When the oligonucleotide is in the hairpin conformation the quencher is capable of substantially suppressing the fluorescence of the flourescent dye until the oligonucleotide is fully unfolded and is not in the hairpin conformation.
Preferably, x, y, and z each are xe2x80x94H, HOxe2x80x94, HOxe2x80x94P(xe2x95x90O)(xe2x80x94OH)xe2x80x94Oxe2x80x94, HOP(xe2x95x90O)(xe2x80x94OH)xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OH)xe2x80x94Oxe2x80x94, HOP(xe2x95x90O)(xe2x80x94OH)xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OH)xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OH)xe2x80x94Oxe2x80x94, xe2x80x94OCH3, xe2x80x94F, xe2x80x94NH2, xe2x80x94Oxe2x80x94allyl, benzyl, dimethoxytrityl, trityl, acetyl, phosphothioate such as is (HO)2P(xe2x95x90S)xe2x80x94Oxe2x80x94 or (HO)2xe2x80x94P(xe2x95x90O)xe2x80x94Oxe2x80x94(HO)P(xe2x95x90O)xe2x80x94Oxe2x80x94(HO)P(xe2x95x90S)xe2x80x94Oxe2x80x94, methyl phosphoamidite, methyl Dhosphonoamidite, H-phosphonate, phosphotriester, xe2x80x94O-propargyl, silyl, and phosphoamidite such as ((CH3)2CH)2xe2x80x94Nxe2x80x94P(OCH2CH2CN)xe2x80x94Oxe2x80x94 or ((CH3)2CH)2xe2x80x94Nxe2x80x94P(OCH3)xe2x80x94Oxe2x80x94, or bonded to a support such as xe2x80x94Oxe2x80x94C(O)xe2x80x94(CH2)2xe2x80x94C(O)-alkylamino-support.
The quencher is preferably a non-fluorescent dye, e.g., acid alizarin violet N, acid black 24, acid blue 29, acid blue 92, acid blue 113, acid blue 120, and blue 161, acid orange 8, acid orange 51, acid orange 74, acid red 1, acid red 4, acid red 8, acid red 37, acid red 40, acid red 88, acid red 97, acid red 106, acid red 151, acid red 183, acid violet 7, acid yellow 17, acid yellow 25, acid yellow 29, acid yellow 34, acid yellow 38, acid yellow 40, acid yellow 42, acid yellow 65, acid yellow 76, acid yellow 99, alizarin yellow 66, crocein orange G, alizarin blue black B, palatine chrome black 6BN, mordant black 3, basic red 29, basic blue 66, brilliant yellow chrysophine, chrysoldin, crocein orange G, crystal scarlet, fast black K salt, fast corinth V salt, fast garnet GBC, fat brown B, fat brown RR, mordant blue 9, mordant brown 1, mordant brown 4, mordant brown 6, mordant brown 24, mordant brown 33, mordant brown 48, mordant orange 1, mordant orange 6, mordant orange 10, oil red E6N, oil red O, orange 11, orange G, palatine chrome black 6BN, palatine fast yellow BLN, tropaeolin O, acid yellow 36, 4-dimethylamino-2-methylazobenzene, disperse orange 1, disperse orange 3, disperse orange 13, disperse orange 25, disperse red 1, disperse red 13, disperse red 19, disperse yellow 3, disperse yellow 5, disperse yellow 7, ethyl red, methyl red, napthyl red, 4-phenylazoaniline, 4-phenylazomalcinanil, 4-phenylazophenol, 4(4-nitrophenolazo)resorcinol, nitro red, orange II, para red, Sudan I, Sudan II, Sudan III, Sudan IV, Sudan Orange G, or Sudan Red 7B; or a non-fluorescent triphenyl methane dye, e.g., alkali blue 6B, aniline blue, aurintricarboxcylic acid, basic violet 14, basic red 9, brilliant green, bromochlorophenol blue, bromocresol purple, chlorophenol red, crystal violet, ethyl violet, fast green FCF, guinea green B, malachite green, methyl green, new fuchsia, pyrocatechol violet, thymol blue, thymolphthalin, Victoria blue B, victoria blue R, or victoria pure blue BO. Preferably, the non-fluorescent triphenyl methane dye contains an isocyanate group, an isothiocyanate group, or an N-succinimidyl ester group. If the quencher is malachite green, it preferably contains an amine group bonded to the atom at the 4 position. Other preferred quenchers are uniblue B, alizarin blue black B, alizarin red S, alizarin violet 3R, fast blue BB, and fast blue RR.
Ideally, the quencher has the following general structure (B): 
wherein T is xe2x80x94NHxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(S)xe2x80x94NHxe2x80x94, or xe2x80x94C(S)xe2x80x94Oxe2x80x94; a, axe2x80x2, b, bxe2x80x2, c, cxe2x80x2, d, dxe2x80x2, and exe2x80x2 are each xe2x80x94H, xe2x80x94Cl, xe2x80x94Br, xe2x80x94F, xe2x80x94OCH3, xe2x80x94OH, xe2x80x94CH3, xe2x80x94SO3xe2x80x94, diazophenyl, diazo-1-naphthyl, xe2x80x94CO2R, or xe2x80x94N(R)2; and R is xe2x80x94H, xe2x80x94CH3, xe2x80x94CH2xe2x80x94CH3, or O.
The invention is also directed to a method for producing a compound containing at least two nucleotide groups, e.g., an oligonucleotide, wherein the method comprises linking the compound of claim 1 to another compound containing at least one nucleotide or nucleotide group.
The compounds of the invention can be readily prepared by the following process:
(a) reacting a pyrimidine nucleotide with a mercuric salt in a suitable solvent under suitable condition so as to form a pyrimidine mercurated at the C-5 position;
(b) reacting said mercurated pyrimidine with a linker having a reactive terminal double or triple bond to react with the -Hg portion of the pyrimidine compound in the presence of K2PdCl4 in an aqueous solvent and under suitable conditions so as to form the linker-pyrimidine compound.
(c) reacting the Pyr-L-NH2 with a quencher, selected from non-fluorescent azo dyes and non-fluorescent triphenyl methane dyes and other suitable compounds having at least one moiety capable of reacting with the amino functionality of the Pyr-L-NH2 preferably with a quencher having a sulfonyl chloride, carboxylic acid chloride, N-succinimidyl ester, isocyanate or isothiocyanate. Alternatively the terminal xe2x80x94NH2 group of the linker is activated by forming an isocyanate or isothiocyanate and then reacting the isocyanate or isothiocyanate with an alcohol or amino moiety on the dye to form the desired pyrimidine-quencher compound.
The P-L-quencher described above is then used in the preparation of primers. The P-L-quencher are readily incorporated into the growing oligonucleotide during its synthesis using automated oligonucleotide techniques and equipment. It was also discovered that DABSYL was a preferred quencher. However, DABSYL or other quenchers are not available in the form of dabsyl-nucleotide phosphoamidites. Consequently, a deoxyuridine xcex2-cyanoethyl phosphoramidite containing a DABSYL moiety linked to the base via a linker arm (dU-dabsyl) was prepared as described in the present application. A number of hairpin primers incorporating a pyrimidine-quencher were synthesized on an automatic DNA synthesizer. Analytical HPLC revealed an increase in primer yields to between 80 to 90%, indicating surprisingly efficient coupling of the dU-DABSYL and stability of the dU-DABSYL through many synthetic cycles. Analysis of the fluorescent properties of these primers revealed unexpectedly high signal to noise ratios in samples that had only been desalted and not purified by HPLC. The enhanced signal to noise in impure samples may be due to the incorporation of the DABSYL before the addition of the fluorophore because the synthesis of oligonucleotides occur in the 3xe2x80x2 to 5xe2x80x2 direction. Consequently, even the most efficient synthesis will have a slight excess of oligonucleotides containing DABSYL with no fluorophore. When the DABSYL is not incorporated into the primer the fluorophore is not quenched when the primer is in the hairpin conformation. This situation is far more likely to occur with the post synthetic modification of the primer using activated dye since the reaction with the amine typically has an efficiency less than 80 percent. An unexpected advantage of incorporation of the quencher molecule by means of a dU-quencher in the probe synthesis is that the probe will have a statistically greater change of having a quencher molecule than a fluorescent dye molecule so that the background fluorescence of the probe is substantially reduced.